Efficient vaccine production requires the growth of large quantities of virus produced in high yields from a host system. Different types of virus require different growth conditions in order to obtain acceptable yields. The host in which the virus is grown is therefore of great significance. As a function of the virus type, a virus may be grown in primary tissue culture cells, established cell lines or in embryonated eggs, such as those from chickens.
The cultivation conditions under which a virus strain is grown also are of great significance with respect to achieving an acceptably high yield of the strain. Thus, in order to maximize the yield of a desired virus strain, both the host system and the cultivation conditions must be adapted specifically to provide an environment that is advantageous for the production of a desired virus strain. Therefore, in order to achieve an acceptably high yield of the various virus strains, a system which provides optimum growth conditions for a large number of different viruses is required. Many viruses are restricted to very specific host systems, some of which are very inefficient with regard to virus yields.
Some of the mammalian cells which are used as viral host systems produce virus at high yields, but the tumorigenic nature of such cells invokes regulatory constraints against their use for vaccine production. In fact, the applicable guidelines of the World Health Organization (WHO) indicate that only a few cell lines are allowed for virus vaccine production.
The problems arising from the use of serum in cell culture and/or protein addivites derived from an animal or human source added to the culture medium, i.e. the varying quality and composition of diffent batches and the risk of contamination with mycoplasma, viruses or BSE-agent, are well-known. In general, serum or serum-derived substances like albumin, transferrin or insulin may contain unwanted agents that can contaminate the culture and the biological products produced therefrom. Furthermore, human serum derived additives have to be tested for all known viruses, like hepatitis or HIV, which can be transmitted by serum. Bovine serum and products derived thereform, for example trypsin, bear the risk of BSE-contamination. In addition, all serum-derived products can be contaminated by still unknown agents. Therefore, many attempts are being made to provide efficient host systems and cultivation conditions that do not require serum or other serum derived compounds.
Over time, many viruses change their serotypes. Any change in virus serotype requires a corresponding change in a vaccine intended to elicit immunity toward the new virus serotype. To maintain the efficiency of the protection accorded by a vaccine to a particular new virus serotype, a new vaccine must be produced which confers immunity to that new serotype. To produce the new vaccine, the new virus strains must be grown. Because many viruses, in particular Influenza virus, change serotype very quickly, the cultivation system must be able to produce viral antigen, including virions, in large-scale quantities sufficiently fast to permit production of vaccines during the infection season of the virus.
In many cases, the optimum growth conditions for the new virus strains are different from the conditions employed to grow their predecessors. Accordingly, a cultivation system that can be easily adjusted to provide the requirements for optimum growth of new virus strains is highly desirable. Moreover, practical considerations, such as the need for high production output of the new strain, render highly desirable a method that is applicable to large scale production of the virus, such as influenza.
One typical example of a virus that changes its serotype frequently is Influenza virus. Influenza is a major respiratory disease in man and is responsible for many thousands of deaths every year.
There are three general types of Influenza viruses, Type A, Type B and Type C. The types are defined by the absence of serological crossreactivity between their internal proteins. Influenza Type A viruses are further classified into sub-types based on antigenic differences of their glycoproteins, the hemagglutinin (HA) and neuraminidase (NA) proteins. Humans are susceptible mainly to diseases caused by infection with Influenza Type A, B, and C viruses.
Currently, the most significant causes of Influenza infections in humans are those attributable to Type B and to subtypes H1N1 and H3N2 of Influenza type A. Accordingly, antigens of Type B and of subtypes H1N1 and H3N2 of Influenza Type A are those which are generally incorporated into present Influenza vaccines. The vaccines currently available have protection rates ranging from 75-90%.
The Influenza HA antigen is the major target for the protective immune responses of a host to the virus. One of the problems in the development of effective influenza vaccines stems from the high mutation rate of the gene coding for the HA protein, resulting in frequent changes in its antigenicity. Therefore, in order to produce effective vaccines, new vaccines from recent Influenza isolates must be produced frequently.
The normal practice of recovering new viral isolates involves recovery with a throat swab or similar source, followed by cultivation of the isolates in embryonated chicken eggs. Although the initial isolation into eggs may be difficult, the virus adapts to its egg host, and large scale production of the virus can be carried out in eggs.
Conventional methods for producing influenza vaccine have always involved the growth of the viruses in embryonated chicken eggs. Viruses grown by this method are then used for producing live attenuated virus, killed whole virus or subunit vaccines. However, conventional methodology involving embryonated chicken eggs to produce influenza vaccine is extremely cumbersome, involving the handling of many thousands of eggs per week. In a typical operation, eggs must be candled, the shell must be sterilized and each egg must be inoculated by injection of a small volume of virus into the allantoic cavity. The injected eggs are then incubated for 48-72 hours at 33.degree.-37.degree. C., candled again, refrigerated overnight and opened to allow harvesting of the allantoic fluid. The harvested fluid must then be clarified by filtration and/or centrifugation before processing for further purification. Extensive purification is then required to ensure freedom from egg protein. Requirements For Inactivated Influenza Vaccine, World Health Organization Technical Report Series, 384 (1966).
In a typical chicken embryo operation, between one and two eggs are required to produce one dose of influenza vaccine. Thus, to produce a million doses of vaccine, more than a million egg embryos must be processed. In summary, the conventional approach to producing influenza virus vaccines involves many steps which are difficult to automate and are, accordingly, labor intensive, time consuming, expensive and subject to contamination. A need therefore exists for methods which are less labor intensive, require less biological tissue per dose produced and are less susceptible to contamination.
There have been many attempts to adapt standard tissue culture technology with primary chicken embryo cells ("CEC") or established mammalian cell lines for Influenza virus vaccine production. These attempts were unsuccessful because a large number of viral strains do not replicate in conventional cultures. The use of established mammalian cell lines, such as Madin-Darby canine kidney (MDCK) line, has been more successful in replicating some strains. Nevertheless, a number of virus strains will not replicate in the MDCK line. In addition, fears over possible adverse effects associated with employing cells with a tumorigenic potential for human vaccine production have precluded the use of MDCK, a highly transformed cell line, in this context.
One of the primary difficulties in growing a number of influenza strains in primary tissue culture or established cell lines arises from the necessity for proteolytic cleavage activation of the influenza hemagglutinin in the host cell. Cleavage of the virus HA.sub.0 precursor into the HA 1 and HA 2 subfragments is a necessary step in order for the virus to infect a new cell. Thus, cleavage is required in order to convert new virus particles in the host cells into virions capable of infecting new cells. Cleavage is known to occur during transport of the integral HA.sub.0 membrane protein from the endoplasmic reticulum of the infected cell to the plasma membrane. In the course of transport, hemagglutinin undergoes a series of co- and post-translational modifications including proteolytic cleavage of the precursor HA into the amino-terminal fragment HA 1 and the carboxyterminal HA 2.
The fact that Influenza virions have been found which contain either uncleaved or cleaved HA glycoproteins indicates that cleavage is not always necessary for virus assembly and release from the infected cell. Cleavage of HA is indeed necessary, however, for the initiation of infection of a new host cell.
Although it is known that an uncleaved HA can mediate attachment of the virus to its neuramic acid-containing receptors at the cell surface, it is not capable of the next step in the infectious cycle, which is fusion. It has been reported that exposure of the hydrophobic amino terminus of the HA 2 by cleavage is required so that it can be inserted into the target cell, thereby forming a bridge between virus and target cell membrane. This is followed by fusion of the two membranes and entry of the virus into the target cell.
Proteolytic activation of hemagglutinin follows a pattern observed with many enzymes and hormone precursors, such as proinsulin, progastrin and proopiomelanocortin. It involves cleavage at an arginine residue by a trypsin-like endoprotease. The available evidence suggests that the endoprotease is an intracellular enzyme which is calcium dependent and has a neutral pH optimum. However, beyond these observations, little is known about the nature of the intracellular protease (Klenk et al, "The Molecular Biology of Influenza Virus Pathogenicity", Adv. Virus Res., 34:247-281 (1988)).
Since the activating proteases are cellular enzymes, the infected cell type determines whether the Influenza hemagglutinin is cleaved. The hemagglutinins of the mammalian Influenza viruses and the nonpathogenic avian Influenza viruses are susceptible to proteolytic cleavage only in a restricted number of cell types. On the other hand, the hemagglutinins of pathogenic avian viruses among the H 5 and H 7 subtypes are cleaved by proteases present in a broad range of different host cells. Thus, there are differences in host range resulting from differences in hemagglutinin cleavability which can be correlated with the pathogenic properties of the virus.
The differences in cleavability are due to differences in the amino acid sequence of the cleavage site of the hemagglutinin. Sequence analyses have revealed that the HA1 and HA2 fragments of the hemagglutinin molecule of the non-pathogenic avian and of all mammalian Influenza viruses are linked by a single arginine. In contrast, the pathogenic avian strains have a sequence of several basic amino acids at the cleavage site with the common denominator being lysine-arginine or arginine-arginine. The hemagglutinins of all Influenza viruses are cleaved by the same general mechanism resulting in the elimination of the basic amino acids.
The protease activities which are essential for cleavage of a broad range of influenza virus strains are available in the embryonated egg and in cell aggregates representing the whole chicken embryo. Conventional CEC cultures prepared from chick embryos, however, provide only some of the protease activities of a whole chicken embryo and, hence, allow replication of a limited range of influenza virus strains. Standard procedures for preparation of CEC cultures involve removal of the head and inner organs and multiple trypsinization steps. These procedures result specifically in the loss of brain, heart, lung, liver and kidney cells, which have been shown to replicate a number of influenza strains (Scholtissek et al., "Multiplication of Influenza A Viruses with Cleavage and Non-cleavable Hemagglutinin in Chicken Embryo Membranes or Organes, and Cell Cultures Derived Therefrom", J. Gen. Virol., 69, 2155-2164 (1988). Standard procedures thus result in a highly selected cell population consisting mainly of fibroblasts, which are limited in terms of the virus strains that they can support.
Improvements in influenza virus production have been attempted before. For instance, it has been reported that the limited replication of several Influenza A strains in standard cell cultures could be ameliorated by the addition of trypsin to the tissue culture medium. For example, trypsin addition significantly increases the infectivity of various strains grown in CEC cultures (Lazarowitz et al., "Enhancement of the Infectivity of Influenza and B Viruses by Proteolytic Cleavage of the Hemagglutinin Polypeptide", Virology, 68:440-454 (1975)). In addition Stieneke-Grober et al., "Influenza Virus Hemagglutinin with Multibasic Site is Activated by Furin, a Subtilisin-like Endoprotease", EMBO J., 11: 2407-2414 (1992), have identified the HA activating enzyme in MDBK cells as a furin-like protease. Such enzymes have been isolated from human and mouse tissues and constitute a new family of eukaryotic subtilisin-like endoproteases.
Other attempts at developing alternative vaccine production methods have been undertaken. U.S. Pat. No. 4,783,411, to Gabliks discusses a method for preparing influenza vaccines in goldfish cell cultures. The virus particles for infecting the Gabliks cultures after their establishment were obtained from chicken embryo cultures or from infected CD-1 strain mice. The virus is passaged at least twice in such goldfish cell cultures, resulting in an attenuated virus which may be used as a live vaccine.
U.S. Pat. No. 4,500,513 to Brown et al. discloses the production of unmodified virus particles for making vaccine from liquid cell culture or cell monolayer culture wherein a protein hydrolyzing enzyme, such as trypsin, chymotrypsin or carboxypeptidase, is incubated with a partially infected culture to increase the proportion of additional cells infected by the virus and to ensure the maximum cytopathic effect. Harvesting of the virus is performed at a point in the growth phase of the virus which exhibits maximum cytopathic effect. All of the examples of Brown, however, describe a dog kidney cell line which is not usable for human vaccine production. Due to the maximum cytopathic effects of the virus in the method according to Brown et al., virus yield is limited to only one round of virus replication. Moreover, Brown does not teach manipulation of the virus genome nor optimization of culture conditions. Therefore, the method of Brown is not applicable for the large-scale production of virus, which is necessary for the efficient production of corresponding vaccines.
U.S. Pat. No. 4,205,131 to Almeida discloses a method for propagating rotavirus in cell culture in the presence of serum-free medium containing the proteolytic enzyme trypsin. Due to the lethal effect on the cells of trypsin at higher levels, the virus yield of Almeida, like Brown, was limited to that produced in one round of replication.
More recently, others have attempted to produce influenza virus in cell-line cultures. For example, Katz et al., J. Infect. Dis. 160:191-98 (1989) has compared the growth characteristics of influenza in MDCK cells and amniotic cavity of embryonated eggs. Katz found that the influenza titer obtained from MDCK cells compared favorably to embryonated eggs. There are problems with using MDCK cells, however. For example, MDCK cells are no licensed cell line for production of human vaccines. Moreover, the Katz procedure requires viruses to be multiply and serially passaged in the MDCK cell line, which is costly and, more importantly, time consuming.
Kaverin et al., J. Virol. 69: 2700-03 (1995) have attempted to grow influenza virus in VERO cells grown in serum-containing medium, VERO cells are licensed by the World Health Organization for general vaccine production.
Kaverin encountered difficulties in propagating influenza virus in VERO cells, however, and linked these difficulties to a loss of trypsin activity in the cell cultures caused by a factor apparently released by the VERO cells. Kaverin addressed this problem by repeatedly adding trypsin, and serial passaging the viruses in the VERO cells. Only after 10 passages in VERO cells did Kaverin obtain a Liter that was as high as could be obtained with embryonated egg and MDCK cells. Similar results were obtained by Govorkova et al., J. Infect. Dis. 172: 250-53 (1995).
Neither Kaverin nor Govorkova address the problems of the use of serum-containing medium, however. Serum-containing mediums generally lack batch-to-batch consistency, and contain undesired contaminants that complicate the viral production and purification process.
These contaminants include contaminating viruses, such as BVDV, Bluetongue virus, prions or BSE, and/or immunogenic proteins, which can present serious safety concerns.
Use of serum-free medium to grow viruses also has been attempted in the prior art. See EP 115442, U.S. Pat. Nos. 4,525,349, 4,664,912. In these methods, the host cells are first grown in serum-containing medium and, just prior to infection with the respective virus, the serum-containing medium is replaced by serum-free medium.
VERO cells have been adapted to growth in serum-free medium, such as MDSS2. Merten et al., Cytotech. 14: 47-59 (1994). MDSS2 lacks growth factors, but still has a significant presence of non-serum proteins (30-40 mg protein/ml). Merten et al., Biologicals 23: 185-89 (1995). Accordingly, MDSS2 is not entirely free of the problems associated with other protein-containing mediums, such as serum-containing mediums.
A continuing need exists for safe and effective methods to produce viruses and their antigen, as well as recombinant proteins in virus-based expression systems. Moreover, there is need for an approach to viral propagation, employing materials that are readily available and requiring a minimal number of time-consuming manipulations, such as adaptation of a virus to a particular cell substrate by serial passaging, that can meet applicable regulatory standards and still accommodate many different viruses and virus strains, especially thoses that can not be multiplied efficiently via conventional methods.